Flourescene energy transfer-based single molecule/ensemble dna sequencing by synthesis

ABSTRACT

This invention provides nucleotide analogues each of which comprises a tag comprising one or more Forster resonance energy transfer (FRET) acceptor fluorophores, a nucleotide polymerase having one or more FRET donor fluorophores, and methods for sequencing single-stranded

Throughout this application, certain publications are referenced, the byauthors and publication year. Full citations for these publications maybe found immediately preceding the claims. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to describe more fully the state of theart to which this invention relates.

BACKGROUND OF THE INVENTION

High throughput DNA sequencing is essential to a broad array of genomicstudies, such as whole genome and metagenome sequencing, expressionprofiling of mRNAs and miRNAs, discovery of alternatively spliced andpolyadenylated transcripts, histone and chromatin changes involved inepigenetic events, and identification of binding sites for transcriptionfactors and RNA binding proteins. Sequencing of individual human genomesis especially appealing, with its potentially unlimited but as yetunachieved promise for personalized medicine.

Given the ever-growing importance of high throughput DNA sequencing forbiological and anthropological research, agriculture and medicine, thereis a need for sequencing technologies that are low-cost and rapid on theone hand, and have high sensitivity and accuracy on the other.Sequencing by Synthesis (SBS) has driven much of the “next generation”sequencing technology, allowing the field to approach the $100,000Genome [Fuller et al. 2009, Hawkins et al. 2010, Morozova et al. 2009,and Park 2009]. With further improvements in nucleotide incorporationdetection methods, SBS could be an engine that drives third-generationplatforms leading to the reality of the “$1,000 Genome”.

Current commercial next-generation sequencing platforms have certainlymade substantial inroads in this direction, with the current cost ofsequencing a human genome at high draft coverage significantly below$10,000 [Fuller et al. 2009, Hawkins et al. 2010, Morozova et al. 2009,and Metzker 2010]. Expression studies (e.g. using RNA-Seq) andepigenetic studies (e.g. using Methyl-Seq, ChIP-Seq), among many others,have also benefited greatly from these platforms (Ozsolak et al. 2011,Varley et al. 2010, and Park 2009). Nonetheless, these costs are stillprohibitive for most laboratories and for clinical applications.

All of the current approaches have one or more additional limitations:biased coverage of GC-rich or AT-rich portions of genomes; inability toaccurately sequence through homopolymer stretches; inability to directlysequence RNA; high reagent costs; difficulty in sequencing beyond 200 orso nucleotides resulting in difficulty in de novo assembly of previouslyunsequenced genomes; insufficient throughput due to ceiling on number ofpossible reads per run.

To overcome these obstacles, a number of third-generation sequencingplatforms have appeared on the market, or are in development. All ofthese have issues with accuracy and most have limited throughput.

The underlying photophysical principle for this SBS method is based onFörster resonance energy transfer (FRET), where the energy of electronicexcited states of a donor molecule is transferred to an acceptormolecule via non-radiative dipole-dipole interactions. As a result, theluminescence of the donor molecule is quenched and fluorescence of theacceptor molecule is observed. The occurrence and efficiency of FRETdepends on various parameters, such as the distance (<10 nm) between thedonor and acceptor as well as the spectral overlap between the donorluminescence and acceptor absorption spectra (Hung et al 1996, Turro etal 2010). In the past FRET has been used for Sanger sequencing (Ju etail 1995). In the current application, this has been extend to SBS.

SUMMARY OF THE INVENTION

A method for determining the identity of a nucleotide in asingle-stranded DNA comprising:

-   a) contacting a composition comprising a single-stranded DNA having    a primer hybridized to a portion thereof, with one or more    non-catalytic metal ions, a nucleotide polymerase, and a nucleotide    analogue having the structure:

-   -   wherein the base is A, C, G, T, or U, or analogues thereof,        wherein X is CH₂, NH, CHF, or CF₂, wherein n is 0, 1, 2, 3, or        4, wherein the acceptor dye is one or more fluorophores, and        wherein X prevents a nucleotide polymerase from hydrolyzing the        bond between the α and β phosphates,    -   under conditions permitting the nucleotide polymerase to form a        ternary complex with the single-stranded DNA, primer, and the        nucleotide analogue if the nucleotide analogue has a base that        is complementary to a nucleotide residue of the single-stranded        DNA which is immediately 5′ to a nucleoside residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer,    -   wherein the DNA polymerase has attached, incorporated, and/or        conjugated fluorescence donor molecules, wherein the donor        molecules are Förster Resonance Energy Transfer (FRET) donors,        and the acceptor dyes on the nucleotide analogue are        corresponding FRET acceptors,    -   wherein if the base of the nucleotide analogue is not        complementary to the nucleotide residue of the single-stranded        DNA which is immediately 5′ to the nucleoside residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, iteratively repeating the contacting with        a different nucleotide analogue until a nucleotide analogue is        complementary to the nucleotide residue of the single-stranded        DNA which is immediately 5′ to the nucleoside residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, thus forming a ternary complex,    -   with the proviso that (i) the type of base on each nucleotide        analogue is different from the type of base on each of the other        nucleotide analogues, and (ii) the acceptor dyes of each        nucleotide analogue fluorophore has a predetermined fluorescent        wavelength emission;

-   b) excite the DNA polymerase donor fluorescent molecules using an    appropriate spectral emission, thereby causing the corresponding    FRET acceptors, which are the acceptor dye organic fluorophores    attached to nucleotide analogue in the ternary complex, to generate    the predetermined fluorescent wavelength emission, and thereby    determine the identity of the nucleotide analogue.

This invention also provides a method for determining the nucleotidesequence of a single-stranded DNA comprising:

-   a) contacting the single-stranded DNA, wherein the single-stranded    DNA has a primer hybridized to a portion thereof, with a    non-catalytic metal ion, a DNA polymerase, and a nucleotide analogue    having the structure:

-   -   wherein the base is A, G, C, T, or U, or analogues thereof,        wherein n is 0, 1, 2, 3, or 4, wherein the acceptor dye is one        or more organic fluorophores, wherein R′ is a cleavable linker        bound to a blocking moiety, and wherein cleaving the linker        results in a 3′-OH,    -   under conditions permitting the DNA polymerase to form a ternary        complex with the single-stranded DNA, primer, and a nucleotide        analogue wherein the nucleotide analogue has a base that is        complementary to a nucleotide residue of the single-stranded DNA        which is immediately 5′ to a nucleoside residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, and wherein the nucleotide polymerase has        attached or incorporated fluorescence donor molecules,    -   wherein the DNA polymerase has attached, incorporated, and/or        conjugated fluorescence donor molecules, wherein the donor        molecules are Förster Resonance Energy Transfer (FRET) donors,        and the acceptor dyes and the acceptor dyes on the nucleotide        analogue are corresponding FRET acceptors;

-   b) exciting the DNA polymerase donor fluorescent molecules using an    appropriate spectral emission, thereby causing the corresponding    FRET acceptors, which are the acceptor dye organic fluorophores    attached to the nucleotide analogue in the ternary complex, to    generate the unique predetermined fluorescent wavelength emission,    and thereby determine the identity of the nucleotide analogue;

-   c) contact the ternary complex with catalytic metal ions permitting    the DNA polymerase to incorporate the nucleotide analogue into the    primer;

-   d) cleave the linker bound to the blocking moiety of the    incorporated analogue thereby resulting in a 3′-OH; Acceptor dye

-   e) iteratively performing steps a) through d) for each nucleotide    residue of the single-stranded DNA to be sequenced so as to thereby    determine the sequence of the single-stranded DNA.

This invention also provides a real-time method for determining thenucleotide sequence of a single-stranded DNA comprising:

-   a) contacting a composition comprising a single-stranded DNA which    has a primer hybridized to a portion thereof, a nucleotide    polymerase, and four nucleotide analogues having the structure:

-   -   wherein the base is A, G, C, T, or U, or analogues thereof,        wherein n is 0, 1, 2, 3, or 4, wherein the acceptor dye is one        or more fluorophores,    -   under conditions permitting the nucleotide polymerase to        incorporate a nucleotide analogue when the nucleotide analogue        has a base that is complementary to a nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, and wherein the nucleotide        polymerase has attached or incorporated fluorescence donor        molecules,    -   wherein the nucleotide polymerase has attached, incorporated,        and/or conjugated fluorescence donor molecules, wherein the        donor molecules are Förster Resonance Energy Transfer (FRET)        donors, and the acceptor dyes and the acceptor dyes on the        nucleotide analogue are corresponding FRET acceptors;

-   b) exciting the nucleotide polymerase fluorescence donor molecules    using an appropriate spectral emission, thereby causing the    corresponding FRET acceptors attached to the nucleotide analogues,    to generate the unique predetermined fluorescent wavelength    emission, and thereby determine the identity of the nucleotide    analogue;

-   c) iteratively performing steps a) through b) for each nucleotide    residue of the single-stranded DNA to be sequenced so as to thereby    determine the sequence of the single-stranded DNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Formation of a ternary complex by the polymerase carrying energydonors near the active site with nucleotides labeled with acceptorfluorophores coupled with primed template enables efficient energytransfer from donor to acceptor that will be detected for sequencing. Toachieve this, repositioning of cysteine residues in the large fragmentof DNA pol I for attachment of these donors was performed. a) Amino acidpositions for attachment of energy donors such as organic fluorophores,quantum dots or metal complexes to the large fragment DNA polymerase Ifrom Geobacillus kaustophilus were selected outside the enzyme'scatalytic center. The cysteine at position C388 in a wild-type enzymewas substituted with lysine and the amino acids in the designatedpositions were in turn substituted with cysteines by means ofrecombinant DNA techniques and can now be used for attachment to donorsalong with C845. b) Distances of 4 to 5 nm between the wild type C845and the various substituted designated cysteine positions ensure thatdonor will be close to the active center of the enzyme. In this way theywill be able to transfer energy to the acceptor dye(s) on the incomingnucleotide. The donor-acceptor distance will be ˜2-4 nm which isfavorable for FRET.

FIG. 2: FRET-based SBS with donor dyes bound to polymerase, acceptordyes bound to nucleotide, and template attached to magnetic heads.Reaction is carried out in solution in microscopic chambers. The donordye(s) is attached to the polymerase. Acceptor dyes are bound to thebase and/or terminal phosphate of natural or reversibly blockednucleotides. The arrows in the figure indicate excitation of the donor,FRET between donor and acceptor, and acceptor emission. With primer ortemplate molecules bound to magnetic beads to restrict localization ofthe resulting complex and permit washing, sequencing reactions can becarried out in both single molecule or ensemble mode (see Examples 1 and2 in text for details). DNA can also be attached directly to the chambersurface.

FIG. 3: Set-up similar to FIG. 2 except that primer Is attached tomagnetic beads or chamber surface. This permits single-moleculesequencing.

FIG. 4: FRET-based SAS with donor dyes bound to polymerase, acceptordyes bound to nucleotide, and polymerase bound to magnetic beads. Thebiotin on the polymerase permits attachment to streptavidin beadsallowing washing. Nucleotides in solution are beyond the Försterresonance distance that will give a detectable acceptor emission signal.The arrows in the figure indicate excitation of the donor, FRET betweendonor and acceptor, and acceptor emission. This set-up is designed forsingle molecule real time sequencing provided each of the fournucleotides has a different acceptor dye or FRET pair. DNA can also beattached directly to the chamber surface.

FIG. 5: Examples of fluorescent labeled non-incorporable nucleotides.Acceptor fluorophores can be attached at terminal phosphate (A), base(B) and or to both terminal phosphate and base (C).

FIG. 6: Examples of 3′-O-reversibly blocked dNTPs for fluorescentsequencing.

FIG. 7: General structure of fluorescently labeled 3′-reversibly blockednucleotide with acceptor fluorophore(s) attached at the terminalphosphate.

FIG. 8: Example of a combinatorial fluorescence energy transfer label onthe terminal phosphate of a 3′-reversibly blocked nucleotide.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a nucleoside polyphosphate analogue having thestructure:

wherein the base is adenine, guanine, cytosine, uracil, thymine, or aderivative thereof, wherein X is CH₂, NH, CHF, or CF₂, wherein n is 0,1, 2, 3, or 4, wherein each acceptor dye is a fluorophore, and where R′is H or a cleavable linker bound to a blocking moiety.

In another embodiment the acceptor dye is 1, 2, or 3 fluorophores. Inanother embodiment the fluorophores are organic fluorophores. In afurther embodiment the organic fluorophores are separated by aseparation distance that prevents the organic fluorophores fromsignificantly quenching each other. In yet a further embodiment theorganic fluorophores are one or more of a cyanine dye, a rhodamine dye,fluorescein, acridine, coumarin, Texas Red dye, BODIPY, GFP, rhodol,ROX, resorfuin, Alexa Flour, Tokyo Green,N,N,N′,N′-tetramethyl-6-carboxyrhodamine, or any derivative thereof.

In another embodiment the cleavable linker is photo-cleavable orchemically cleavable. In another embodiment the cleavable linker is anyone of an allyl group, alkyl group, carbonyl group, Sieber linkers,indole, disulfide, dithiomethyl group, azidomethyl group, nitrobenzylgroup.

This invention also provides method for determining the identity of anucleotide in a single-stranded DNA comprising:

-   a) contacting a composition comprising a single-stranded DNA having    a primer hybridized to a portion thereof, a nucleotide polymerase,    and a nucleotide analogue having the structure:

-   -   wherein the base is A, C, G, T, or U, or analogues thereof,        wherein X is CH₂, NH, CHF, or CF₂, wherein n is 0, 1, 2, 3, or        4, wherein the acceptor dye is one or more fluorophores, and        wherein X prevents a nucleotide polymerase from hydrolyzing the        bond between the α and β phosphates,    -   under conditions permitting the nucleotide polymerase to form a        ternary complex with the single-stranded DNA, primer, and the        nucleotide analogue if the nucleotide analogue has a base that        is complementary to a nucleotide residue of the single-stranded        DNA which is immediately 5′ to a nucleoside residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer,    -   wherein the DNA polymerase has attached, incorporated, and/or        conjugated fluorescence donor molecules, wherein the donor        molecules are Förster. Resonance Energy Transfer (FRET) donors,        and the acceptor dyes on the nucleotide analogue are        corresponding FRET acceptors,    -   wherein if the base of the nucleotide analogue is not        complementary to the nucleotide residue of the single-stranded        DNA which is immediately 5′ to the nucleoside residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, iteratively repeating the contacting with        a different nucleotide analogue until a nucleotide analogue is        complementary to the nucleotide residue of the single-stranded        DNA which is immediately 5′ to the nucleoside residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, thus forming a ternary complex,    -   with the proviso that (1) the type of base on each nucleotide        analogue is different from the type of base on each of the other        nucleotide analogues, and (ii) the acceptor dyes of each        nucleotide analogue fluorophore has a predetermined fluorescent        wavelength emission;

-   b) exciting the DNA polymerase donor fluorescent molecules using an    appropriate spectral emission, thereby causing the corresponding    FRET acceptors, which are the acceptor dye organic fluorophores    attached to nucleotide analogue in the ternary complex, to generate    the predetermined fluorescent wavelength emission, and thereby    determine the identity of the nucleotide analogue.

This invention also provides a method for determining the nucleotidesequence of a single-stranded DNA comprising:

-   a) contacting the single-stranded DNA which has a primer hybridized    to a portion thereof, a nucleotide polymerase, and a nucleotide    analogue having the structure:

-   -   wherein the base is A, G, C, T, or U, or analogues thereof,        wherein X is CH₂, NH, CHF, or CF₂, wherein n is 0, 1, 2, 3, or        4, wherein the acceptor dye is a fluorophore, and wherein X        prevents a nucleotide polymerase from hydrolyzing the bond        between the α and β phosphates    -   wherein the nucleotide polymerase has attached, incorporated,        and/or conjugated fluorescence donor molecules, wherein the        donor molecules are Förster Resonance Energy Transfer (FRET)        donors, and the acceptor dyes on the nucleotide analogue are        corresponding FRET acceptors,    -   under conditions permitting the DNA polymerase to form a ternary        complex with the single-stranded DNA, primer, and nucleotide        analogue if the analogue has a base that is complementary to a        nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleoside residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the        primer,    -   and if the base of the nucleotide analogue is not complementary        to the nucleotide residue of the single-stranded DNA which is        immediately 5′ to the nucleoside residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the        primer, iteratively repeating the contacting with a different        nucleotide analogue until the analogue is complementary to the        nucleotide residue of the single-stranded DNA which is        immediately 5′ to the nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the        primer, thus forming a ternary complex, with the proviso        that (i) the type of base on each nucleotide analogue is        different from the type of base on each of the other nucleotide        analogues, and (ii) the fluorophore of each nucleotide analogue        has a predetermined fluorescent wavelength emission;

-   b) excite the nucleotide polymerase donor fluorescent molecules    using an appropriate spectral emission, thereby causing the    corresponding FRET acceptors, which are the acceptor dye organic    fluorophores attached to the nucleotide analogue in the ternary    complex, to generate the predetermined fluorescent wavelength    emission, and thereby determine the identity of the nucleotide    analogue;

-   c) contact the ternary complex with 3′-O blocked nucleotide    reversible terminators under conditions permitting the nucleotide    polymerase to catalyze incorporation onto the primer of a    3′O-blocked nucleotide reversible terminator complementary to a    nucleotide residue of the single-stranded DNA which is immediately    5′ to a nucleotide residue of the single-stranded DNA hybridized to    the 3′ terminal nucleotide residue of the primer, thereby replacing    the nucleotide analogue in the ternary complex, wherein the 3′-O    blocked nucleotide reversible terminators have the structure:

-   -   wherein R′ is a cleavable linker bound to a blocking moiety, and        wherein cleaving the linker results in a 3′-OH;

-   d) cleaving the linker bound to the blocking moiety of the    incorporated 3′-O blocked nucleotide reversible terminator, thereby    resulting in a 3′-OH:

-   e) iteratively performing steps a) through d) for each nucleotide    residue of the single-stranded DNA to be sequenced so as to thereby    determine the sequence of the single-stranded DNA.

This invention also provides a method for determining the nucleotidesequence of a single-stranded DNA comprising:

-   a) contacting a composition comprising a single-stranded DNA which    has a primer hybridized to a portion thereof, a nucleotide    polymerase, and four nucleotide analogues having the structure:

-   -   wherein the base is adenine, guanine, cytosine, uracil, thymine,        or analogues thereof, wherein n is 0, 1, 2, 3, or 4, wherein the        acceptor dye is one or more fluorophores, and wherein X prevents        a nucleotide polymerase from hydrolyzing the bond between the α        and β phosphates,    -   wherein the nucleotide polymerase has attached, incorporated,        and/or conjugated fluorescence donor molecules, wherein the        donor molecules are Förster. Resonance Energy Transfer (FRET)        donors, and the acceptor dyes on the nucleotide analogue are        corresponding FRET acceptors,    -   wherein (i) the type of base on each analogue is different from        the type of base on each of the other three analogues, (ii) the        fluorophores of each nucleotide analogue have a unique        predetermined fluorescent wavelength emission that corresponds        to the type of base, and (iii) the fluorophores of each analogue        are FRET acceptors that are excited by same donor fluorescence        molecules in the nucleotide polymerase;    -   under conditions permitting the nucleotide polymerase to form a        ternary complex with the single-stranded DNA, primer, and a        nucleotide analogue wherein the nucleotide analogue has a base        that is complementary to a nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleoside        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer,

-   b) exciting the nucleotide polymerase fluorescent donor molecules    using an appropriate spectral emission, thereby causing the    corresponding FRET acceptors, which are the acceptor dye    fluorophores attached to the nucleotide analogue in the ternary    complex, to generate the unique predetermined fluorescent wavelength    emission, and thereby determine the identity of the nucleotide    analogue;

-   c) contact the ternary complex with 3′-O blocked nucleotide    reversible terminators under conditions permitting the nucleotide    polymerase to catalyze incorporation onto the primer of a    3′O-blocked nucleotide reversible terminator complementary to a    nucleotide residue of the single-stranded DNA which is immediately    5′ to a nucleotide residue of the single-stranded DNA hybridized to    the 3′ terminal nucleotide residue of the primer, thereby replacing    the nucleotide analogue in the ternary complex, wherein the 3′-O    blocked nucleotide reversible terminators have the structure:

-   -   wherein R′ is a cleavable linker bound to a blocking moiety, and        wherein cleaving the linker results in a 3′-CH;

-   d) cleaving the linker bound to the blocking moiety of the    incorporated 3′-O blocked nucleotide reversible terminator, thereby    resulting in a 3′-OH;

-   e) iteratively performing steps a) through d) for each nucleotide    residue of the single-stranded DNA to be sequenced so as to thereby    determine the sequence of the single-stranded DNA.

This invention also provides a real-time method for determining thenucleotide sequence of a single-stranded DNA comprising:

-   -   a) contacting a composition comprising a single-stranded DNA        which has a primer hybridized to a portion thereof, a nucleotide        polymerase, and four nucleotide analogues having the structure:

-   -   wherein the base is A, G, C, T, or U, or analogues thereof,        wherein n is 0, 1, 2, 3, or 4, wherein the acceptor dye is one        or more fluorophores,    -   under conditions permitting the nucleotide polymerase to        incorporate a nucleotide analogue when the nucleotide analogue        has a base that is complementary to a nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, and wherein the nucleotide        polymerase has attached or incorporated fluorescence donor        molecules,    -   wherein the nucleotide polymerase has attached, incorporated,        and/or conjugated fluorescence donor molecules, wherein the        donor molecules are Förster Resonance Energy Transfer (FRET)        donors, and the acceptor dyes and the acceptor dyes on the        nucleotide analogue are corresponding FRET acceptors;

-   b) exciting the nucleotide polymerase fluorescence donor molecules    using an appropriate spectral emission, thereby causing the    corresponding FRET acceptors attached to the nucleotide analogues,    to generate the unique predetermined fluorescent wavelength    emission, and thereby determine the identity of the nucleotide    analogue;

-   c) iteratively performing steps a) through b) for each nucleotide    residue of the single-stranded DNA to be sequenced so as to thereby    determine the sequence of the single-stranded DNA.

In a further embodiment the acceptor dye is 2, or 3 organicfluorophores. In a further embodiment the organic fluorophore is acyanine dye, a rhodamine dye, fluorescein, acridine, coumarin, Texas Reddye, BODIPY, GFP, rhodol, ROX, resorfuin, Alexa Flour, quantum dot,Tokyo Green, N,N,N′,N′-tetramethyl-6-carboxyrhodamine, or In a furtherembodiment the polymerase fluorescence donor molecules are one or moreof a cyanine dye, a rhodamine dye, fluorescein, acridine, coumarin,Texas Red dye, BODIPY, GFP, rhodol, ROX, resorfuin, Alexa Flour, aquantum dot, Tokyo Green, an Ru(II) polypyridyl complex,N,N,N′,N′-tetramethyl-6-carboxyrhodamine, or any derivative thereof.

In a further embodiment the cleavable linker is photo-cleavable orchemically cleavable.

In a further embodiment the cleavable linker is any one of an allylgroup, alkyl group, carbonyl group, Sieber linkers, indole, disulfide,dithiomethyl, azidomethyl, nitrobenzyl group.

In a further embodiment the cleavable linker is cleaved using Pd(0),tetrabutylammonium, OTT, a triphosphine, peroxydisulphate, iodine, orany derivative thereof.

In a further embodiment optionally a buffer wash occurs after each ofsteps a), b), c), and/or d).

In a further embodiment the primer or single-stranded DNA are bound to amagnetic bead or the surface of a fluidic chamber.

In a further embodiment the polymerase the primer or single-stranded DNAare bound to the magnetic bead or surface are modified with one ofamino, sulfhydryl, or biotin moieties.

In a further embodiment, the single-stranded DNA is amplified usingemulsion PCR thereby resulting in a plurality of copies of thesingle-stranded DNA.

In a further embodiment the method is simultaneously performed on theplurality of single-stranded DNA copies.

In another embodiment prior to step a), several copies of thesingle-stranded DNA are created on a bead using emulsion PCT.

In another embodiment prior to step a), several copies of thesingle-stranded DNA are created on a surface using bridge amplification.

In another embodiment the single-stranded DNA is bound to a surface andremains there during the iterative process.

In another embodiment prior to step a) catalytic metal ions are removed.

In another embodiment the acceptor dye is 1, 2, or 3 organicfluorophores.

In another embodiment the organic fluorophores are separates: by aseparation distance that prevents the organic fluorophores fromsignificantly quenching each other.

In an embodiment when the ternary complex is formed, the nucleotidepolymerase fluorescence donor molecule and the nucleoside polyphosphateanalogue acceptor fluorophore are less than 10 nm from each other.

In an embodiment the nucleotide polymerase fluorescence donor moleculeand the nucleoside polyphosphate analogue acceptor fluorophore arebetween 2 nm-4 nm from each other.

In an embodiment time-gated luminescence detection techniques are usedto detect the nucleoside polyphosphate analogue acceptor emissionsignal.

In an embodiment the nucleotide polymerase is a mutant Geobacilluskaustophilus DNA polymerase I or Phi29 DNA polymerase.

In an embodiment the polymerase has pairs of cysteines in antipodallocations wherein the fluorescence donor molecules are attached.

This invention also provides a method for determining the nucleotidesequence of a single-stranded DNA comprising:

-   a) contacting a composition comprising a single-stranded DNA,    wherein the single-stranded DNA has a primer hybridized to a portion    thereof, with a non-catalytic metal ion, a nucleotide polymerase,    and a nucleotide analogue having the structure:

-   -   wherein the base is A, G, C, T, or U, or analogues thereof,        wherein n is 0, 1, 2, 3, or 4, wherein the acceptor dye is one        or more fluorophores, wherein R′ is a cleavable linker bound to        a blocking moiety, and wherein cleaving the linker results in a        3′-OH,    -   wherein the nucleotide polymerase has attached, incorporated,        and/or conjugated fluorescence donor molecules, wherein the        donor molecules are Förster resonance Energy Transfer (FRET)        donors, and the acceptor dyes on the Nucleotide analogue are        corresponding FRET acceptors,    -   wherein (i) the type of base on each analogue is different from        the type of base on each of the other three analogues, (ii) the        fluorophores of each nucleotide analogue have a unique        predetermined fluorescent wavelength emission that corresponds        to the type of base, and (iii) the fluorophores of each analogue        are FRET acceptors that are excited by same donor fluorescence        molecules in the nucleotide polymerase;    -   under conditions permitting the nucleotide polymerase to form a        ternary complex with the single-stranded DNA, primer, and a        nucleotide analogue wherein the nucleotide analogue has a base        that is complementary to a nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleoside        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer;

-   b) exciting the nucleotide polymerase fluorescence donor molecules    using an appropriate spectral emission, thereby causing the    corresponding FRET acceptors, which are the acceptor dye    fluorophores attached to the nucleotide analogue in the ternary    complex, to generate the unique predetermined fluorescent wavelength    emission, and thereby determine the identity of the nucleotide    analogue;

-   c) contacting the ternary complex with catalytic; metal ions    permitting the nucleotide polymerase to incorporate the nucleotide    analogue into the primer;

-   d) cleaving the linker bound to the blocking moiety of the    incorporated analogue thereby resulting in a 3′-OH:

-   e) iteratively performing steps a) through d) for each nucleotide    residue of the single-stranded DNA to be sequenced so as to thereby    determine the sequence of the single-stranded DNA.

This invention also provides a method for determining the nucleotidesequence of a single-stranded DNA comprising:

-   a) contacting a composition comprising a single-stranded DNA,    wherein the single-stranded DNA has a primer hybridized to a portion    thereof, with a non-catalytic metal ion, a nucleotide polymerase,    and four nucleotide analogues having the structure:

-   -   wherein the base is A, G, C, T, or U, or analogues thereof,        wherein n is 0, 1, 2, 3, or 4, wherein the acceptor dye is one        or more fluorophores, wherein R′ is a cleavable Linker bound to        a blocking moiety, and wherein cleaving the linker results in a        3′-OH,    -   wherein the nucleotide polymerase has attached, incorporated,        and/or conjugated fluorescence donor molecules, wherein the        donor molecules are Förster Resonance Energy Transfer (FRET)        donors, and the acceptor dyes on the nucleotide analogue are        corresponding FRET acceptors,    -   under conditions permitting the nucleotide polymerase to form a        ternary complex with the single-stranded DNA, primer, and a        nucleotide analogue wherein the nucleotide analogue has a base        that is complementary to a nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleoside        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer,

-   b) exciting the nucleotide polymerase fluorescence donor molecules    using an appropriate spectral emission, thereby causing the    corresponding FRET acceptors, which are the acceptor dye    fluorophores attached to the nucleotide analogue in the ternary    complex, to generate the unique predetermined fluorescent wavelength    emission, and thereby determine the identity of the nucleotide    analogue;

-   c) contacting the ternary complex with catalytic metal ions    permitting the nucleotide polymerase to incorporate the nucleotide    analogue into the primer;

-   d) cleaving the linker bound to the blocking moiety of the    incorporated analogue thereby resulting in a 3′-OH;

-   e) iteratively performing steps a) through d) for each nucleotide    residue of the single-stranded DNA to be sequenced so as to thereby    determine the sequence of the single-stranded DNA.

In a further embodiment the acceptor dye is 2, or 3 organicfluorophores. In a further embodiment the organic fluorophore is acyanine dye, a rhodamine dye, fluorescein, acridine, coumarin, Texas Reddye, BODIPY, GFP, rhodol, ROX, resorfuin, Alexa Flour, quantum dot,Tokyo Green, N,N,N′,N′-tetramethyl-6-carboxyrhodamine, or In a furtherembodiment the polymerase fluorescence donor molecules are one or moreof a cyanine dye, a rhodamine dye, fluorescein, acridine, coumarin,Texas Red dye, BODIPY, GFP, rhodol, ROX, resorfuin, Alexa Flour, aquantum dot, Tokyo Green, an Ru(II) polypyridyl complex,N,N,N′,N′-tetramethyl-6-carboxyrhodamine, or any derivative thereof.

In a further embodiment the cleavable linker is photo-cleavable orchemically cleavable.

In a further embodiment the cleavable linker is any one of an allylgroup, alkyl group, carbonyl group, Sieber linkers, indole, disulfide,dithiomethyl, azidomethyl, nitrobenzyl group.

In a further embodiment the cleavable linker is cleaved using Pd(0),tetrabutylammonium, DTT, a triphosphine, peroxydisulphate, iodine, orany derivative thereof.

In a further embodiment optionally a buffer wash occurs after each ofsteps a), b), c), and/or d).

In a further embodiment the primer or single-stranded DNA are bound to amagnetic bead or the surface of a fluidic chamber.

In a further embodiment the polymerase the primer or single-stranded DNAare bound to the magnetic bead or surface are modified with one ofamino, sulfhydryl, or biotin moieties.

In a further embodiment, the single-stranded DNA is amplified usingemulsion PCR thereby resulting in a plurality of copies of thesingle-stranded DNA.

In a further embodiment the method is simultaneously performed on theplurality of single-stranded DNA copies.

In another embodiment prior to step a), several copies of thesingle-stranded DNA are created on a bead using emulsion PCT.

In another embodiment prior to step a), several copies of thesingle-stranded DNA are created on a surface using bridge amplification.

In another embodiment the single-stranded DNA is bound to a surface andremains there during the iterative process.

In another embodiment prior to step a) catalytic metal ions are removed.

In another embodiment the acceptor dye is 1, 2, or 3 organicfluorophores.

In another embodiment the organic fluorophores are separated by aseparation distance that prevents the organic fluorophores fromsignificantly quenching each other.

In an embodiment when the ternary complex is formed, the nucleotidepolymerase fluorescence donor molecule and the nucleoside polyphosphateanalogue acceptor fluorophore are less than 10 nm from each other.

In an embodiment the nucleotide polymerase fluorescence donor moleculeand the nucleoside polyphosphate analogue acceptor fluorophore arebetween 2 nm-4 nm from each other.

In an embodiment time-gated luminescence detection: techniques are usedto detect the nucleoside polyphosphate analogue acceptor emissionsignal.

In an embodiment the nucleotide polymerase is a mutant Geobacilluskaustophilus DNA polymerase I or Phi29 DNA polymerase.

In an embodiment the polymerase has pairs of cysteines in antipodallocations wherein the fluorescence donor molecules are attached.

In an embodiment the non-catalytic metal ions are Ca⁺⁺ and/or Sr++.

In an embodiment the catalytic metal ions are Mg⁺⁺ and/or Mn⁺⁺.

For the foregoing embodiments, each embodiment disclosed herein iscontemplated as being applicable to each of the other disclosedembodiments. In addition, the elements recited in the nucleotideanalogue embodiments can be used in the composition and methodembodiments described herein and vice versa.

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

-   -   A—Adenine;    -   C—Cytosine;    -   G—Guanine;    -   T—Thymine;    -   U—Uracil;    -   DNA—Deoxyribonucleic acid;    -   RNA—Ribonucleic acid;

“Nucleic acid” shall mean, unless otherwise specified, any nucleic acidmolecule, including, without limitation, DNA, RNA and hybrids thereof.In an embodiment the nucleic acid bases that form nucleic acid moleculescan be the bases A, C, G, T and U, as well as derivatives thereof.Derivatives of these bases are well known in the art, and areexemplified in. PCR Systems, Reagents and Consumables (Perkin ElmerCatalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J.,USA).

“Substrate” or “Surface” shall mean any suitable medium present in thesolid phase to which a nucleic acid or an agent may be affixed.Non-limiting examples include chips, beads, nanopore structures andcolumns. In an embodiment the solid substrate can be present in asolution, including an aqueous solution, a gel, or a fluid.

“Hybridize” shall mean the annealing of one single-stranded nucleic acidto another nucleic acid based on the well-understood principle ofsequence complementarity. In an embodiment the other nucleic acid is asingle-stranded nucleic acid. The propensity for hybridization betweennucleic acids depends on the temperature and ionic strength of theirmilieu, the length of the nucleic acids and the degree ofcomplementarity. The effect of these parameters on hybridization is wellknown in the art (see Sambrook J, Fritsch E F, Maniatis T. 1989.Molecular cloning: a laboratory manual. Cold Spring Harbor LaboratoryPress, New York). As used herein, hybridization of a primer sequence, orof a DNA extension product, to another, nucleic acid shall meanannealing sufficient such that the primer, or DNA extension product,respectively, is extendable by creation of a phosphodiester bond with anavailable nucleotide or nucleotide analog capable of forming aphosphodiester bond.

As used herein, unless otherwise specified, a base which is “differentfrom” another base or a recited list of bases shall mean that the basehas a different structure from the other base or bases. For example, abase that is “different from” adenine, thymine, and cytosine wouldinclude a base that is guanine or a base that is uracil.

As used herein, unless otherwise specified, a tag moiety which isdifferent from the tag moiety of a referenced molecule means that thetag moiety has a different chemical structure from the chemicalstructure of the other/referenced tag moiety.

In certain embodiments the underlying photophysical principle for thisSAS method is based on Förster resonance energy transfer (FRET), wherethe energy of electronic excited states of a donor molecule istransferred to an acceptor molecule via non-radiative dipole-dipoleinteractions. As a result, the luminescence of the donor molecule isquenched and fluorescence of the acceptor molecule is observed. Theoccurrence and efficiency of FRET depends on various parameters, such asthe distance (<10 nm) between the donor and acceptor as well as thespectral overlap between the donor luminescence and acceptor absorptionspectra (Hung et al 1996, Turro et al 2010

In certain embodiments, the polymerase, single-stranded polynucleotide,RNA, or primer is bound to a solid substrate via 1,3-dipolarazide-alkyne cycloaddition chemistry. In an embodiment the polymerase,DNA, RNA, or primer, is bound to the solid substrate via a polyethyleneglycol molecule. In an embodiment the polymerase, DNA, RNA, primer, orprobe is alkyne-labeled. In an embodiment the polymerase, DNA, RNA,primer, or probe is bound to the solid substrate via a polyethyleneglycol molecule and the solid substrate is azide-functionalized. In anembodiment the polymerase, DNA, RNA, or primer, is immobilized on thesolid substrate via an azido linkage, an alkynyl linkage, orbiotin-streptavidin interaction. Immobilization of nucleic acids isdescribed in Immobilization of DNA on Chips II, edited by ChristineWittmann (2005), Springer Verlag, Berlin, which is hereby incorporatedby reference. In an embodiment the DNA is single-strandedpolynucleotide. In an embodiment the RNA is single-stranded RNA.

In other embodiments, the solid substrate is in the form of a chip, abead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, aporous media, a porous nanotube, or a column. This invention alsoprovides the instant method, wherein the solid substrate is a metal,gold, silver, quartz, silica, a plastic, polypropylene, a glass, ordiamond. This invention also provides the instant method, wherein thesolid substrate is a porous non-metal substance to which is attached orimpregnated a metal or combination of metals. The solid surface may bein different forms including the non-limiting examples of a chip, abead, a tube, a matrix, a nanotube. The solid surface may be made frommaterials common for DNA microarrays, including the non-limitingexamples of glass or nylon. The solid surface, for examplebeads/micro-beads, may be in turn immobilized to another solid surfacesuch as a chip.

In various embodiments the polymerase, nucleic acid samples, DNA, RNA,primer, or probe are separated in discrete compartments, wells ordepressions on a surface.

In this invention methods are provided wherein about 1000 or fewercopies of the polymerase, nucleic acid sample, DNA, RNA, or primer arebound to the substrate. This invention also provides the instant methodswherein 2×10⁷, 1×10⁷, 1×10⁶ or 1×10⁴ or fewer copies of the polymerase,nucleic acid sample, DNA, RNA, or primer are bound to the substrate orsurface.

In some embodiments, the immobilized polymerase, nucleic acid sample,DNA, RNA, or primer, is immobilized at a high density. This inventionalso provides the instant methods wherein over or up to 1×10⁷, 1×10⁸,1×10³ copies of the polymerase, nucleic acid sample, DNA, RNA, or primerare bound to the substrate or surface.

In other embodiments of the methods and/or compositions of thisinvention, the DNA is single-stranded. In other embodiments of themethods or of the compositions described herein, the single-strandedpolynucleotide is replaced with an RNA that is single-stranded.

In certain embodiments, UV light is used to photochemically cleave thephotochemically cleavable linkers and moieties. In an embodiment, thephotocleavable linker is a 2-nitrobenzyl moiety.

A “nucleotide residue” is a single nucleotide in the state it existsafter being incorporated into, and thereby becoming a monomer of, apolynucleotide. Thus, a nucleotide residue is a nucleotide monomer of apolynucleotide, e.g. DNA, which is bound to an adjacent nucleotidemonomer of the polynucleotide through a phosphodiester bond at the 3′position of its sugar and is bound to a second adjacent nucleotidemonomer through its phosphate group, with the exceptions that (I) a 3′terminal nucleotide residue is only bound to one adjacent nucleotidemonomer of the polynucleotide by a phosphodiester bond from itsphosphate group, and (ii) a 5′ terminal nucleotide residue is only boundto one adjacent nucleotide monomer of the polynucleotide by aphosphodiester bond from the 3′ position of its sugar.

Because of well-understood base-pairing rules, determining the FRETenergy electronic excited states of the nucleoside polyphosphateanalogue incorporated into a primer or DNA extension product, andthereby the identity of the dNTP analog that was incorporated, permitsidentification of the complementary nucleotide residue in thesingle-stranded polynucleotide that the primer or DNA extension productis hybridized to. Thus, if the dNTP analog that was incorporated has aunique wavenumber in the Raman spectroscopy peak identifying it ascomprising an adenine, a thymine, a cytosine, or a guanine, then thecomplementary nucleotide residue in the single-stranded polynucleotideis identified as a thymine, an adenine, a guanine or a cytosine,respectively. The purine adenine (A) pairs with the pyrimidine thymine(T). The pyrimidine cytosine (C) pairs with the purine guanine (G).Similarly, with regard to RNA, if the dNTP analog that was incorporatedcomprises an adenine, a uracil, a cytosine, or a guanine, then thecomplementary nucleotide residue in the single-stranded RNA isidentified as a uracil, an adenine, a guanine or a cytosine,respectively.

Incorporation into an oligonucleotide or polynucleotide (such as aprimer or DNA extension strand) of a nucleotide and/or nucleoside analogmeans the formation of a phosphodiester bond between the 3′ carbon atomof the 3′ terminal nucleotide residue of the polynucleotide and the 5′carbon atom of the dNTP analog resulting in the loss of pyrophosphatefrom the dNTP analog.

Where a range of values is provided, unless the context clearly dictatesotherwise, it is understood that each intervening integer of the value,and each tenth of each intervening integer of the value, unless thecontext clearly dictates otherwise, between the upper and lower limit ofthat range, and any other stated or intervening value in that statedrange, is encompassed within the invention. The upper and lower limitsof these smaller ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding (i) either or (ii)both of those included limits are also included in the invention.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and may be unsubstituted or substituted. Thus, C1-Cn as in“C1-Cn alkyl” includes groups having 1, 2, . . . , n−1 or n carbons in alinear or branched arrangement. For example, a “C1-C5 alkyl” includesgroups having 1, 2, 3, 4, or 5 carbons in a linear or branchedarrangement, and specifically includes methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, and pentyl.

As used herein, “alkenyl” refers to a non-aromatic hydrocarbon group,straight or branched, containing at least 1 carbon to carbon doublebond, and up to the maximum possible number of non-aromaticcarbon-carbon double bonds may be present, and may be unsubstituted orsubstituted. For example, “C2-C5 alkenyl” means an alkenyl group having2, 3, 4, or 5, carbon atoms, and up to 1, 2, 3, or 4, carbon-carbondouble bonds respectively. Alkenyl groups include ethenyl, propenyl, andbutenyl.

The term “alkynyl” refers to a hydrocarbon group straight or branched,containing at least 1 carbon to carbon triple bond, and up to themaximum possible number of non-aromatic carbon-carbon triple bonds maybe present, and may be unsubstituted or substituted. Thus, “C2-C5alkynyl” means an alkynyl group having 2 or 3 carbon atoms and 1carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl andbutynyl.

The term “substituted” refers to a functional group as described abovesuch as an alkyl, or a hydrocarbyl, in which at least one bond to ahydrogen atom contained therein is replaced by a bond to non-hydrogen ornon-carbon atom, provided that normal valencies are maintained and thatthe substitution(s) result(s) in a stable nucleotide analogue.Substituted groups also include groups in which one or more bonds to acarbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Non-limiting examplesof substituents include the functional groups described above, and forexample, N, e.g. so as to form —CN.

It is understood that substituents and substitution patterns on thenucleotide analogues of the instant invention can be selected by one ofordinary skill in the art to provide nucleotide analogues that arechemically stable and that can be readily synthesized by techniquesknown in the art, as well as those methods set forth below, from readilyavailable starting materials. If a substituent is itself substitutedwith more than one group, it is understood that these multiple groupsmay be on the same carbon or on different carbons, so long as a stablestructure results.

In choosing the nucleotide analogues of the present invention, one ofordinary skill in the art will recognize that the various substituents,i.e. R₁, R₂, etc. are to be chosen in conformity with well-knownprinciples of chemical structure connectivity.

In the nucleotide analogue structures depicted herein, hydrogen atoms,except on ribose and deoxyribose sugars, are generally not shown.However, it is understood that sufficient hydrogen atoms exist on therepresented carbon atoms to satisfy the octet rule.

All combinations of the various elements described herein are within thescope of the invention. All sub-combinations of the various elementsdescribed herein are also within the scope of the invention.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS

Disclosed herein is a FRET based sequencing by synthesis (SBS) approach,in which a polymerase molecule is conjugated to 1 or more fluorescencedonor molecules (fluorophores at a higher energy/lower wavelength than aparticular acceptor fluorophore). The positions selected for attachmenthave the following properties: (1) they do not interfere with thepolymerase enzymatic function, i.e., they should be excluded from thekey binding pockets and active center of the enzyme as well as otheramino acids required for enzyme activity; and (2) they are distributedover the enzyme surface so as to produce a localized FRET between thedonor fluorophores on the polymerase and the acceptor fluorophores onthe incoming nucleotide. FRET typically acts at a distance in the rangeof 1-10 nm. Given that most polymerases have dimensions in the 4-10 nmrange, placement of 1-3 donor molecules should accomplish thisobjective, allowing the donor to be localized within 3 nm of theacceptor-labeled nucleotide. Disclosed herein is substantial informationobtained by generating mutants of several DNA polymerases that do notinhibit enzyme activity that are used to further refine these positions.As an example, Geobacillus kaustophilus DNA polymerase 1 mutants wereproduced with pairs of cysteines in various antipodal locations thatcould be used for attachment of energy donor molecules (FIG. 1) such asquantum dots (Nikiforov et al 2010, Peng et al 2012), organicfluorophors, or Ru(II) polypyridyl complexes (Marti et al 2007). Manyadditional positions for attachment of the donor dyes in thispolymerase, and by extension other polymerases, to meet theserequirements. Best positions for placement vary with the intensity ofthe donor emission and the size of the donor molecules. Moreover, thewavelengths for donor emission and acceptor absorbance have to becarefully selected to maximize FRET while minimizing competitiveabsorption. It has been demonstrated that particles as large as quantumdots can be attached to Phi29 DNA polymerase (Nikiforov et al 2010);these could be useful because their photophysical properties can bevaried over a wide range. Quantum dots with light absorption over abroad wavelength range, but with narrow bandpass and high extinctioncoefficients, are available to optimize the donor absorption propertieswith the excitation light source. Luminescence lifetimes in quantum dotsare often longer than most organic fluorophores, which increases theprobability of FRET to the acceptor. Further increase of the donorluminescence lifetime is possible with the use of metal complexes, suchas Ru(II) polypyridyl complexes as donors. Because energy transfer fromthe long-lived metal to ligand charge transfer (MLCT) excited state tosinglet states of organic fluorophores, such as Cy5, is spin-forbidden,this energy transfer is slow, which results in a delayed fluorescence ofthe acceptor fluorophore (Marti et al 2007). Using time-gatedluminescence detection techniques, such as fluorescence lifetimemicroscopy, the background signal originating from light scattering,autofluorescence, and direct excitation of the acceptor fluorophores canbe eliminated.

Two different setups for carrying out SBS using intermolecular FRET aredepicted in FIGS. 2 and 3. In the first, the DNA is linked to magneticbeads or the surface of the fluidic chamber. Either the template (shown)or the primer may be attached to the surface. Many different linkagestrategies are available. For instance the DNA may be modified withamino, sulfhydryl, or biotin moieties and reacted with beads derivatizedwith NHS succinimide, maleimide and streptavidin, respectively. Otherchemical pairs, including but not limited to azide-alkyne andtrans-cyclooctene-tetrazine, are also feasible. Many homobifunctionaland heterobifunctional cross-linkers of assorted lengths arecommercially available, including photoactivatable ones. If instead,adaptors axe attached to the beads, it is possible to amplify the DNA byemulsion PCR to allow ensemble sequencing. In this case, an excess ofpolymerase in solution will interact with many of the templates on thesame bead. In the second setup, the donor dye-decorated polymerases willbe directly conjugated to the magnetic beads or solid surface. Again,many linkage strategies are available in addition to thebiotin-Streptavidin pair shown in FIG. 3.

Since the efficiency of energy transfer is inversely proportional to thesixth power of the distance between donor and acceptor, it is unlikelythat free nucleotides in solution will participate in energy transferand so not contribute to the acceptor emission. Though not the preferredformat, the acceptor dyes can be attached to the polymerase and a donordye to the nucleotide, and would be limited to single color sequencing.

SBS can be performed in one-color mode with the same acceptorfluorophore on each of the four nucleotides. In this case, thesenucleotides are added sequentially one by one in the course of thesequencing reaction. A four-color sequencing mode is possible with theuse of multiple donor acid/or acceptor dyes in conjunction withcombinatorial energy transfer to generate unique fluorescence signaturesfor each of the four nucleotides. Previously, we have demonstrated thatwith one type of donor dye (e.g. fluorescein) and two acceptorfluorophores (e.g. N, N, N′,N′-tetramethyl-6-carboxyrhodamine andcyanine-5) several distinct fluorescence signatures can be generated byvarying the distance between the fluorophores (Ju et al 1995, Hung et al1996, Tong et al 2001).

Herein disclosed are three experimental examples of sequencingapproaches. Experiments 1-2 utilize the setup shown in FIG. 2, whileExperiment 3 uses the setup shown in FIG. 3 (template attached tosurface for single molecule or ensemble sequencing) or FIG. 4 (primerattached to surface for single molecule sequencing). For single moleculemethods, random template libraries are allowed to attach to the beads orfluidic chamber surface individually. Alternatively, for ensemblesequencing, individual DNA molecules may be amplified on the magneticbeads by emulsion PCR or on the surface of the chamber by bridgeamplification. Experiment 3 is a real-time single molecule sequencingapproach.

Experiment 1: FRET-Based SBS with Donor Dye on Polymerase, Acceptor Dyeon Terminal Phosphate and Base of Unincorporable Nucleotides

This SBS reaction uses un-incorporable nucleotides, α, β-X-2′deoxynucleoside 5′-triphosphates (PCP-dNTPs) or polyphosphates(PCP-dNPPs), where X can be CH₂, NH, CHF or CF₂ (Upton et al 2009) andwhere the terminal phosphate and/or the base is derivatized with one tothree acceptor fluorophores, where the separation distance between istuned to avoid self-quenching (FIG. 5). These are added to the reactionchamber containing the bead-bound template (primer), free primer(template), and donor-decorated polymerase, resulting in the formationof a ternary complex consisting of polymerase, template, primer andnucleotide. A magnet placed below the reaction chamber will attract thebeads with the bound ternary complexes to the bottoms of the chambers.This allows for multiple solution changes, and ensures preciselocalization over many cycles of sequencing. Because cleavage of the α,p bond in these nucleotides cannot take place, they are unable to beincorporated into DNA. Thus the ternary complex is monitored forsufficient time to obtain a convincing FRET signal using a TIRF or otherappropriate fluorescent detection device. Excitation of the donor dye atan appropriate wavelength induces energy transfer from the donor to theacceptor fluorophores, generating a unique emission signal for theacceptor dye. Donor-acceptor dye pairs (e.g., high absorptioncross-section cyanine dye as donor with Cy5 as acceptor, quantum dotdonor with ROX acceptor) are selected with some spectral overlap butwhere competitive absorption is minimized. The chamber is then flushedwith a high concentration of unlabeled NRTs. These replace thenon-hydrolyzable phosphate nucleotide in the ternary complex and beincorporated. The NRTs may have any of a variety of blocking groupsattached to the 3′-OH as shown in FIG. 6 such as allyl or azidomethylgroups (Guo et al 2008, Ju et al 2006). Following the addition of theappropriate chemical (Pd(0) or tetrabutylammoniumperoxydisulphate/iodine for allyl, LiBF4 for methoxymethyl (Lipschutz etal 1982, Ireland and Varney 1986), TCEP for azidomethyl and disulfide)or light (in the case of the 2-nitrobenzyl blocker) to reverse theattachment of the blocking group and restore the 3′-OH group, thenon-catalytic metal ions are added back to the system in preparation forthe next cycle, in which the next unincorporable nucleotide is added.Buffer washes are carried out between each reagent addition to reducebackground. As mentioned above, the 3NA can also be attached directly tothe surface of the chamber.

Example 2: FRET-Based SBS with Donor Dye on Polymerase, Acceptor Dye onTerminal Phosphate of Nucleotide Reversible Terminators (NRTs)

In this approach, the initial mixture consists of the magneticbead-bound template or primer DNA, the free primer or template, thedonor dye-bearing polymerase, and NRTs with one or two acceptor dyes onthe terminal phosphate (FIG. 7). In addition the solution containsnon-catalytic metal ions such as Sr⁺⁺ or Ca⁺⁺ (Vander Horn 2014).Briefly, after amplification of DNA on the beads by emulsion PCR ordirectly on the chamber surface by bridge amplification if the ensemblesequencing format is desired, they are washed to remove Mg⁺⁺ and anyother catalytic metal ions. When the beads are deposited onto thesurface, Sr⁺⁺ or Ca⁺⁺ is added along with the primer, polymerase and thenucleotides bearing the 3′ blocking group and the terminalphosphate-bound acceptor dye(s). After sufficient time to form theternary complex, acceptor emission measurements are made. Followingmeasurement for as long as needed to obtain a convincing FRET signature,catalytic metals such as Mg⁺⁺ or Mn⁺⁺ are added to allow incorporation,and after a buffer wash, TCEP is added to remove the blocking group.Finally, Ca⁺⁺ or Sr⁺⁺ is added back to the solution in preparation forthe addition of the subsequent nucleotide to the solution for the nextcycle of the sequencing process. Non-catalytic metals can also be addedbefore the deblocking step to prevent incorporation of any residualnucleotides in the case of homopolymer stretches, but this is unlikelyto be necessary if washes are thorough. The advantages of this methodare that it only requires a set of four nucleotides, and that in eachround it restores the growing DNA strand to a natural DNA helical state.In the case of single molecule sequencing, instead of using templatesamplified on beads, templates may instead be reacted with primers bounddirectly to the surface of the reaction chamber.

Example 3: Single Molecule Real-Time FRET SBS

In this variant, the polymerase is attached to the magnetic beads, andthe template, primer, and nucleotides will be present in the surroundingbuffer. Four different nucleotides with acceptor fluorophores are addedat the same time, each able to be excited by the donor on thepolymerase, but each bearing a different acceptor or FRET pair ofacceptors. An example of a combinatorial fluorescent energy transfer tagis shown in FIG. 8. In this 4-color method, continuous fluorescentmonitoring is required in order to follow the nucleotide incorporationevents in real time. This approach requires the use of a TIRF instrumentor other sophisticated imaging device.

REFERENCES

-   Hung, S-C., et al Cyanine dyes with high absorption cross section as    donor chromophores in energy transfer primers. 243, 15-27 (1996).-   Turro, N. J., Ramamurthy, V., Scaiano, J. C. Modern Molecular    Photochemistry of Organic Molecules, University Science Books,    Sausalito, Calif., USA (2010).-   Ju, J., et al. Fluorescence energy transfer dye-labeled primers for    DNA sequencing and analysis. Proc. Natl. Acad. Sci. U.S.A. 92,    4347-4351 (1995).-   Nikiforov, T., et al. Conjugates of biomolecules to nanoparticles.    U.S. Pat. No. 8,603,792 B2 (2013).-   Peng, Y., et al. CdSe/ZnS core shell quantum dots based FRET binary    oligonucleotide probes for detection of nucleic acids. Photochem.    Photobiol. Sci. 11, 881-884 (2012).-   Marti, A. A., et al. Inorganic-organic hybrid luminescent binary    probe for UNA detection based on spin-forbidden resonance energy    transfer. J. Am. Chem. Soc., 129, 8680-8681 (2007).-   Tong, A. K., et al. Triple energy transfer in covalently    trichromophore-labeled DNA. J. Am. Chem. Soc. 123, 12923-12924    (2001).-   Upton, T. G., et al. Alpha, beta-difluoromethylene deoxynucleoside    5′-triphosphates: a convenient synthesis of useful probes for DNA    polymerase beta structure and function. Org. Lett. 11, 1883-1886    (2009).-   Guo J, et al. Four-color DNA sequencing with 3′-O-modified    nucleotide reversible terminators and chemically cleavable    fluorescent dideoxynucleotides. Proc. Natl. Acad. Sci. U.S.A. 105,    9145-9150 (2008).-   Ju, J., et al. Four-color DNA sequencing by synthesis using    cleavable fluorescent nucleotide reversible terminators. Proc. Natl.    Acad. Sci. U.S.A. 103, 19635-19640 (2006).-   Lipshutz, B. H., Harvey, D. F. Hydrolysis of acetals and ketals    using LiBF4, Synth. Commun. 12, 267-277 (1982).-   Ireland, R. E., Varney M. D. Approach to the total synthesis of    chlorothricolide: synthesis of    (+/−)-19,20-dihydro-24-O-methylchlorothricolide, methyl-ester, ethyl    carbonate. J. Org. Chem. 51, 635-648 (1986).-   Yang, S. G., Park, M. Y., Kim, Y. H. Facile and chemo-selective    cleavages of allyl ethers utilizing tetrabutylammonium sulfate    radical species. Synlett. 2002, 492-994 (2002).-   Vander Horn, P. B. Nucleotide transient binding for sequencing    methods. U.S. Pat. No. 3,863,2975 B2 (2014

1-37. (canceled)
 38. A method for determining the nucleotide sequence ofa single-stranded DNA comprising: a) contacting the single-stranded DNAwhich has a primer hybridized to a portion thereof, a polymerase, and anucleotide analogue, under conditions permitting the polymerase to forma ternary complex with the single-stranded DNA having the primerhybridized thereto and the nucleotide analogue, wherein the nucleotideanalogue has a base that is complementary to a nucleotide residue of thesingle-stranded DNA which is immediately 5′ to a nucleoside residue ofthe single-stranded DNA hybridized to the 3′ terminal nucleotide residueof the primer, wherein the nucleotide analogue is not incorporated bythe polymerase onto the primer, and wherein the nucleotide analogue hasmultiple fluorescent dyes attached thereupon; b) identifying thenucleotide analogue in the ternary complex by detecting the fluorescentemission; c) contacting the ternary complex with 3′-O blocked nucleotidereversible terminators under conditions permitting the nucleotidepolymerase to catalyze incorporation onto the primer of a 3′-O-blockednucleotide reversible terminator complementary to a nucleotide residueof the single-stranded DNA which is immediately 5′ to a nucleotideresidue of the single-stranded DNA hybridized to the 3′ terminalnucleotide residue of the primer, thereby replacing the nucleotideanalogue in the ternary complex; d) cleaving the 3′-O-blocking moiety ofthe incorporated 3′-O blocked nucleotide reversible terminator, therebyresulting in a 3′-OH; and e) iteratively performing steps a) through c)for each nucleotide residue of the single-stranded DNA to be sequencedso as to thereby determine the sequence of the single-stranded DNA. 39.The method of claim 38, wherein the DNA polymerase has attached,incorporated, and/or conjugated fluorescence donor molecules, whereinthe donor molecules are Forster Resonance Energy Transfer (FRET) donors,and the acceptor dyes on the nucleotide analogue are corresponding FRETacceptors.
 40. The method of claim 39, wherein the acceptor dyecomprises 1, 2, or 3 organic fluorophores.
 41. The method of claim 40,wherein the organic fluorophore is a cyanine dye, a rhodamine dye,fluorescein, acridine, coumarin, Texas Red dye, BODIPY, GFP, rhodol,ROX, resorfuin, Alexa Flour, Tokyo Green, N, N,N′,N′-tetramethyl-6-carboxyrhodamine, or a plurality of any of theforegoing.
 42. The method of claim 38, wherein the primer,single-stranded DNA, or nucleotide polymerase are bound to a magneticbead or the surface of a fluidic chamber.
 43. The method of claim 42,wherein the primer, single-stranded DNA, or nucleotide polymerase boundto the magnetic bead or surface are modified with one of amino,sulfhydryl, or biotin moieties.
 44. The method of claim 38, wherein themethod is performed simultaneously on a plurality of single-strandedDNAs.
 45. The method of claim 39, wherein prior to step a), thesingle-stranded DNA is amplified using emulsion PCR thereby resulting ina plurality of copies of the single-stranded DNA.
 46. The method ofclaim 39, wherein when the ternary complex is formed, the nucleotidepolymerase fluorescence donor molecule and the nucleotide analogueacceptor fluorophore are less than 10 nm from each other.
 47. The methodof claim 39, wherein the DNA polymerase fluorescence donor molecule andthe nucleotide analogue acceptor fluorophore are between 2 nm-4 nm fromeach other.
 48. A method for determining the nucleotide sequence of asingle-stranded DNA comprising: a) contacting the single-stranded DNAwhich has a primer hybridized to a portion thereof, a polymerase, and 4nucleotide analogues having the base of A, T, C and G, respectively,under conditions permitting the polymerase to form a ternary complexwith the single-stranded DNA having the primer hybridized thereto andone of the nucleotide analogues, wherein said nucleotide analogue has abase that is complementary to a nucleotide residue of thesingle-stranded DNA which is immediately 5′ to a nucleoside residue ofthe single-stranded DNA hybridized to the 3′ terminal nucleotide residueof the primer, wherein said nucleotide analogue is not incorporated bythe polymerase onto the primer, and wherein each nucleotide analogue hasmultiple fluorescent dyes with distinct emission attached thereto; b)identifying the nucleotide analogue in the ternary complex by detectingits unique fluorescent emission; c) contacting the ternary complex with3′-O blocked nucleotide reversible terminators under conditionspermitting the nucleotide polymerase to catalyze incorporation onto theprimer of a 3′-O-blocked nucleotide reversible terminator complementaryto a nucleotide residue of the single-stranded DNA which is immediately5′ to a nucleotide residue of the single-stranded DNA hybridized to the3′ terminal nucleotide residue of the primer, thereby replacing thenucleotide analogue in the ternary complex; d) cleaving the3′-O-blocking moiety of the incorporated 3′-O blocked nucleotidereversible terminator, thereby resulting in a 3′-OH; and e) iterativelyperforming steps a) through c) for each nucleotide residue of thesingle-stranded DNA to be sequenced so as to thereby determine thesequence of the single-stranded DNA.
 49. The method of claim 48, whereinthe DNA polymerase has attached, incorporated, and/or conjugatedfluorescence donor molecules, wherein the donor molecules are ForsterResonance Energy Transfer (FRET) donors, and the acceptor dyes on thenucleotide analogue are corresponding FRET acceptors.
 50. The method ofclaim 49, wherein the acceptor dye comprises 1, 2, or 3 organicfluorophores.
 51. The method of claim 50, wherein the organicfluorophore is a cyanine dye, a rhodamine dye, fluorescein, acridine,coumarin, Texas Red dye, BODIPY, GFP, rhodol, ROX, resorfuin, AlexaFlour, Tokyo Green, N,N,N′,N′-tetramethyl-6-carboxyrhodamine, or aplurality of any of the foregoing.
 52. The method of claim 48, whereinthe primer, single-stranded DNA, or nucleotide polymerase are bound to amagnetic bead or the surface of a fluidic chamber.
 53. The method ofclaim 52, wherein the primer, single-stranded DNA, or nucleotidepolymerase bound to the magnetic bead or surface are modified with oneof amino, sulfhydryl, or biotin moieties.
 54. The method of claim 48,wherein the method is performed simultaneously on a plurality ofsingle-stranded DNAs.
 55. The method of claim 49, wherein prior to stepa), the single-stranded DNA is amplified using emulsion PCR therebyresulting in a plurality of copies of the single-stranded DNA.
 56. Themethod of claim 49, wherein when the ternary complex is formed, thenucleotide polymerase fluorescence donor molecule and the nucleotideanalogue acceptor fluorophore are less than 10 nm from each other. 57.The method of claim 49, wherein the DNA polymerase fluorescence donormolecule and the nucleotide analogue acceptor fluorophore are between 2nm-4 nm from each other.